Yersinia pestis (Yp) is the causative agent of bubonic and pneumonic plague. The increasing number of plague cases globally (2010-2017), including the U.S., with a ~18% case fatality rate may reflect climate changes and a rodent carrier range shift. An outbreak of 2017 in Madagascar with ~2400 cases (>75% pneumonic) and ~9% causalities has led WHO (April 2018) to intensify the need for developing new generation subunit and live- attenuated plague vaccines. Yp?s ability to persist in dead hosts, and then resurge after years of silence, is alarming. Also, antibiotic-resistant Yp strains occur naturally or have been intentionally developed and currently no FDA-approved plague vaccine is available. Finally, two-component subunit vaccines composed of capsular antigen F1 and a T3SS component and effector LcrV (low calcium response V antigen), which only generate a humoral immune response, provide variable protection in African green monkeys (AGM) and generate poor T cell-mediate immune responses in humans. Since the cellular immunity is also critical for protection, we focused first on identifying new virulence genes of Yp and then to delete such genes in combination to develop novel live-attenuated vaccine strains. Our data indicated that two such vaccine candidates were 100% attenuated in inducing bubonic/pneumonic plague in mice/rats and generated long-term robust humoral and cellular immune responses to provide 100% protection to rodents against developing pneumonic plague. No clinical symptoms of the disease or histopathological lesions were noted either during immunization or when the vaccinated animals were subsequently exposed to wild-type Yp CO92 in a pneumonic plague model. We hypothesize that further immunological characterization of these mutants and their testing in higher animals, such as cynomolgus macaques (CM) and AGM, will provide a rationale for future preclinical and clinical studies. There is a precedent for using a live-attenuated vaccine against plague, as EV76 strain is used in humans in some parts of the world where plague is endemic. However, the vaccine is reactogenic, represents a spontaneous mutant, and causes disease in patients with over iron load. Three Aims have been proposed: Aim 1, to demonstrate efficacy and immune responses of two vaccine candidates generated from Yp CO92 (biovar Orientalis) against other Yp biovars (Antiqua and Medievalis), and the F1-minus mutant of CO92 in bubonic and pneumonic mouse models. Our data with the mutants indicated a potential role of IL-17 (a Th17 cytokine), Th1-IFN-?, and antibodies in protection. In Aim 2, we will study the mechanistic basis of this protection (one chosen mutant) by using ROR?t- /- mice, which lack Th17 cells, as well as IFN-?, and IgA k/o mice and to discern their links to neutrophil recruitment and mucosal immunity, to combat Yp infection in bubonic/pneumonic plague models. In Aim 3, CM and AGM will be used with one mutant to demonstrate its short- and long-term efficacy in causing bubonic and/or pneumonic plague as well as reactogenicity. The correlates of protective immunity will then be established. These innovative mechanistic/translational approaches will result in effective new generation plague vaccines.